US2024044851A1PendingUtilityA1
Method for detecting content of active ingredients of compound sophorae flavescentis radix injection and fingerprint spectrum thereof
Assignee: BEIJING ZHENDONG GUANGMING PHARMACEUTICAL RES INSTITUTE CO LTDPriority: Dec 2, 2020Filed: Nov 30, 2021Published: Feb 8, 2024
Est. expiryDec 2, 2040(~14.3 yrs left)· nominal 20-yr term from priority
G01N 30/34G01N 30/8686G01N 2030/027G01N 30/02G01N 30/06G01N 30/88G01N 2030/884
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Claims
Abstract
A method for detecting the content of active ingredients of a compound Sophorae flavescentis radix injection and the fingerprint spectrum thereof. The method comprises using high performance liquid chromatography to perform detection, wherein the high performance liquid chromatography is operated under the condition of a C18 chromatographic column, and active ingredients comprise matrine, oxymatrine, macrozamin, sophocarpine, oxysophocarpine, sophoridine, or/and piscidic acid. The present method is an improved method for performing detection on a compound Sophorae flavescentis radix injection, by means of same, seven ingredients in the compound Sophorae flavescentis radix injection can be simultaneously determined, and a chromatographic fingerprint spectrum can be constructed, thereby providing a technical method for quality control of a compound Sophorae flavescentis radix injection.
Claims
exact text as granted — not AI-modified1 . A method for detecting contents and fingerprints of active ingredients in Compound Kushen Injection, comprising: performing detection by using a high-performance liquid chromatography, wherein conditions for the high-performance liquid chromatography comprise a C 18 column as a chromatographic column; and the active ingredients comprise matrine, oxymatrine, macrozamin, sophocarpine, oxysophocarpine and sophoridine, or/and 2,3-dihydroxy-2-[(4-hydroxyphenyl)methyl]butanedioic acid.
2 . The method according to claim 1 , wherein the chromatographic column is selected from the group consisting of Waters XSelect CSH™ C 18 , TechMate C 18 -ST, Welch Ultimate AQ-C 18 , and Waters SunFire C 18 , more preferably Waters XSelect CSH™ C 18 , with a dimension of 5 m and 4.6 mm×250 mm.
3 . The method according to claim 2 , wherein the method further comprises a mobile phase comprising methanol in the organic phase and a phosphate buffer gradient elution in the aqueous phase; preferably 0.1%-0.34% potassium dihydrogen phosphate-methanol gradient elution; and more preferably, 0.2% potassium dihydrogen phosphate-methanol gradient elution.
4 . The method according to claim 3 , wherein the pH value of potassium dihydrogen phosphate is adjusted to 2.9-3.1, more preferably to 3.0, with phosphoric acid.
5 . The method according to claim 3 , wherein conditions for gradient elution are:
0.2% potassium
dihydrogen phosphate
(adjusted to pH3.0 with
time (min)
methanol (%)
phosphoric acid) (%)
0-10
3
97
10-15
3-5
97-95
15-24
5-15
95-85
24-30
15
85
30-55
15-85
85-15
55-60
85
15
60-75
3
97
6 . The method according to claim 1 , wherein conditions for the high performance liquid chromatography in the method comprise a column temperature of 28-32° C., preferably 30° C.
7 . The method according to claim 1 , wherein conditions for the high performance liquid chromatography in the method comprise a flow rate of 0.58-0.62 ml/ml, preferably 0.6 ml/min.
8 . The method according to claim 1 , wherein conditions for the high performance liquid chromatography in the method comprise a detection wavelength of 209-213 nm, preferably 211 nm.
9 . The method according to claim 1 , wherein conditions for the high performance liquid chromatography in the method comprise an injection amount of 3-20 μl, preferred 5-15 μl, more preferred 8-12 μl, and most preferably 10 μl.
10 . The method according to claim 1 , wherein conditions for the high performance liquid chromatography in the method comprise preparation of blank solution: adjusting a pH value of potassium dihydrogen phosphate solution to 3.0 with phosphoric acid, preparing a mixed solution of 0.2% potassium dihydrogen phosphate solution-methanol=85:15, and filtering.
11 . The method according to claim 1 , wherein conditions for the high performance liquid chromatography in the method comprise:
preparation of reference substance solution: accurately weighing an appropriate amount of matrine reference substance, oxymatrine reference substance, and oxysophocarpine reference substance, adding the blank solution to prepare a mixed reference substance solution I containing 0.33 mg of matrine, 0.85 mg of oxymatrine, and 0.25 mg of oxysophocarpine per 1 ml, and shaking, which is obtained; accurately weighing an appropriate amount of sophocarpine reference substance, sophoridine reference substance, and macrozamin reference substance, adding the blank solution to prepare a mixed reference substance solution II containing 0.09 mg of sophocarpine, 0.08 mg of sophoridine, and 0.08 mg of macrozamin per 1 ml, and shaking, which is obtained; and accurately weighing 2 ml of the mixed reference substance solution I and II, adding to a 10 ml volumetric flask, diluting with the blank solution to scale, and shaking, which is obtained; or accurately weighing an appropriate amount of 2,3-dihydroxy-2-[(4-hydroxyphenyl)methyl]butanedioic acid reference substance, adding the blank solution to prepare a reference substance stock solution containing 0.25 mg of 2,3-dihydroxy-2-[(4-hydroxyphenyl)methyl]butanedioic acid per 1 ml, and shaking, which is obtained; and accurately weighing 2 ml of the reference substance stock solution, adding to a 10 ml volumetric flask, diluting with the blank solution to scale, and shaking, which is obtained.
12 . The method according to claim 1 , wherein conditions for the high performance liquid chromatography in the method comprise the preparation of the test substance solution: accurately weighing 1 ml of Compound Kushen Injection, adding to a 50 ml volumetric flask, adding a blank solution to scale, shaking, filtering, and taking the subsequent filtrate as the test substance solution.
13 . A method for detecting the content of active ingredients in Compound Kushen Injection according to claim 1 , wherein performing detection by using a high-performance liquid chromatography method, and wherein conditions for the high-performance liquid chromatography comprise:
Detection conditions
Chromatographic
Waters XSelect CSH ™ C18 (5 μm, 4.6 mm × 250 mm)
column
Mobile phase
0.2% Potassium dihydrogen phosphate solution
(adjusted to pH 3.0 with phosphoric acid)-
Methanol gradient elution
Methanol
0.2% Potassium
Time (min)
(%)
dihydrogen phosphate(%)
Elution
0-10
3
97
gradient
10-15
3-5
97-95
15-24
5-15
95-85
24-30
15
85
30-55
15-85
85-15
55-60
85
15
60-75
3
97
Column
30°
C.
temperature
Detection
211
nm
length
Flowing speed
0.6
ml/min
Injection
10
μl
volume
(1) Preparation of a blank solution: adjusting the pH value of potassium dihydrogen phosphate solution to 3.0 with phosphoric acid, preparing a mixed solution of 0.2% potassium dihydrogen phosphate solution-methanol=85:15, and filtering, which is obtained;
(2) Preparation of a reference substance solution: accurately weighing an appropriate amount of matrine reference substance, oxymatrine reference substance, and oxysophocarpine reference substance, adding the blank solution to prepare a mixed reference substance solution I containing 0.33 mg of matrine, 0.85 mg of oxymatrine, and 0.25 mg of oxysophocarpine per 1 ml, and shaking, which is obtained; accurately weighing an appropriate amount of sophocarpine reference substance, sophoridine reference substance, and macrozamin reference substance, adding a blank solution to prepare a mixed reference substance solution II containing 0.09 mg of sophocarpine, 0.08 mg of sophoridine, and 0.08 mg of macrozamin per 1 ml, and shaking, which is obtained; and accurately weighing 2 ml of mixed reference substance solution I and II, adding to a 10 ml volumetric flask, diluting with the blank solution to scale, and shaking, which is obtained; or,
accurately weighing an appropriate amount of 2,3-dihydroxy-2-[(4-hydroxyphenyl)methyl]butanedioic acid reference substance, adding the blank solution to prepare a reference substance stock solution containing 0.25 mg of 2,3-dihydroxy-2-[(4-hydroxyphenyl)methyl]butanedioic acid per 1 ml, and shaking, which is obtained; and accurately weighing 2 ml of the reference substance stock solution, adding to a 10 ml volumetric flask, diluting with the blank solution to scale, and shaking, which is obtained;
(3) Preparation of test substance solution: accurately weighing 1 ml of Compound Kushen Injection, adding to a 50 ml volumetric flask, adding the blank solution to scale, shaking, filtering to obtain a subsequent filtrate as the test substance solution; and
(4) Injecting the blank solution, the reference substance solution, and the test substance solution into the liquid chromatograph in sequence, recording the chromatogram, and calculating the content using an external standard method.
14 . A method for detecting a fingerprint of Compound Kushen Injection according to claim 1 , wherein the method comprises constructing a fingerprint of the Compound Kushen Injection containing matrine, oxymatrine, macrozamin, sophocarpine, oxysophocarpine, and sophoridine.
15 . The method for detecting a fingerprint of Compound Kushen Injection according to claim 14 , wherein the method comprises: performing detection by using a high-performance liquid chromatography, and wherein the conditions for the high-performance liquid chromatography comprise:
Detection conditions
Chromatographic
Waters XSelect CSH ™ C 18 (5 μm, 4.6 mm × 250 mm)
column
Mobile phase
0.2% Potassium dihydrogen phosphate solution
(adjusted to pH 3.0 with
phosporic acid)-Methanol gradient elution
Methanol
0.2% Potassium
Time (min)
(%)
dihydrogen phosphate(%)
Elution
0-10
3
97
gradient
10-15
3-5
97-95
15-24
5-15
95-85
24-30
15
85
30-55
15-85
85-15
55-60
85
15
60-75
3
97
Column
30°
C.
temperature
Detection
211
nm
length
Flowing speed
0.6
ml/min
Injection
10
μl
volume
(1) Preparation of a blank solution: adjusting the pH value of potassium dihydrogen phosphate solution to 3.0 with phosphoric acid, preparing a mixed solution of 0.2% potassium dihydrogen phosphate solution-methanol=85:15, and filtering, which is obtained;
(2) Preparation of reference substance solution: accurately weighing an appropriate amount of matrine reference substance, oxymatrine reference substance, and oxysophocarpine reference substance, adding the blank solution to prepare a mixed reference substance solution I containing 0.33 mg of matrine, 0.85 mg of oxymatrine, and 0.25 mg of oxysophocarpine per 1 ml, and shaking, which is obtained; accurately weighing an appropriate amount of sophocarpine reference substance, sophoridine reference substance, and macrozamin reference substance, adding the blank solution to prepare a mixed reference substance solution II containing 0.09 mg of sophocarpine, 0.08 mg of sophoridine, and 0.08 mg of macrozamin per 1 ml, and shaking, which is obtained; and accurately weighing 2 ml of the mixed reference substance solution I and II, adding to a 10 ml volumetric flask, diluting with the blank solution to scale, and shaking; or
(3) Preparation of test substance solution: accurately weighing 1 ml of the Compound Kushen Injection, adding to a 50 ml volumetric flask, adding the blank solution to scale, shaking, and filtering to obtain a subsequent filtrate as the test substance solution;
(4) Injecting samples in the order of the blank solution, reference substance solution, and the test substance solution to construct a fingerprint of the Compound Kushen Injection containing matrine, oxymatrine, macrozamin, sophocarpine, oxysophocarpine, and sophoridine; and
(5) Detection: injecting samples in the order of the blank solution, the reference substance solution, and the test substance solution to perform detection.
16 . The method according to claim 15 , wherein the fingerprint in step (4) has 10 common characteristic peaks, wherein, based on peak 7-oxymatrine as a reference, the relative retention time of peak 1-sophoramine is 0.442; the relative retention time of peak 2-macrozamin is 0.603; the relative retention time of peak 3-matrine is 0.693; the relative retention time of peak 4-sophocarpine is 0.816; the relative retention time of peak 5-sophoridine is 0.845; the relative retention time of peak 6-oxysophocarpine is 0.941; the relative retention time of peak 7-oxymatrine is 1.0; the relative retention time of peak 8-2,3-dihydroxy-2-[(4-hydroxyphenyl)methyl]butanedioic acid is 1.149; the relative retention time of peak 9 is 1.639; and the relative retention time of peak 10-trifolirhizin is 1.888.Join the waitlist — get patent alerts
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